Obtaining Wisent early blastocyst in vitro is a basic for protection and creation of biodiversity for this threatened species.

Wisent, or European bison (Bison bonasus), is listed as "vulnerable" on the IUCN Red List of Threatened Species and is therefore protected by international law. For the first time, a Wisent embryo has been obtained in vitro. This procedure creates a new opportunity to protect and increase Wisent reproductive potential and thereby opens new possibilities for the establishment of a controlled and broad reserve of the gene pool.

New tregitopes inducing adaptive regulatory T cells in mice.

Epitopes of regulatory T cells (tregitopes) represent linear sequences of amino acids that induce CD4+CD25+Foxp3+ T lymphocytes expansion both in vitro and in vivo. The tregitopes' effectiveness was confirmed in autoimmune disease mouse models and in murine transplant models. Therefore, tregitopes together with regulatory T cells (Tregs) could play a major role in maintaining immune tolerance. The purpose of the presented study was a selection of potential tregitopes and assessment of their impact on Tregs expansion. Eight peptides were selected based on the previously published in silico model and their immunotolerogenic functions. To verify, if selected peptides are potential TCR ligands, the affinity of selected peptides to overrepresented in patients with autoimmune diseases, HLA-DRB1*04:01 allele, was measured by surface plasmon resonance. In order to evaluate the impact of potential tregitopes on the induction of Tregs in in vitro conditions, C57BL6Foxp3GFP mouse antigen presenting cells were co-cultured with naive syngeneic T cells under stimulation of selected peptides. CD4+CD25+Foxp3+ and CD4+CD25+Foxp3+IL-10+ cells frequency was analyzed using flow cytometry. Based on Tregs induction, two tregitopes derived from yeast and adenovirus protein were identified. In summary, the performed studies allowed an identification of novel putative tregitopes, which application potential includes their use as immunomodulators in mice.

Humoral and Cellular Immune Response in Canine Hypothyroidism.

Hypothyroidism is one of the most common endocrine diseases in dogs and is generally considered to be autoimmune in nature. In human hypothyroidism, the thyroid gland is destroyed by both cellular (i.e. autoreactive helper and cytotoxic T lymphocytes) and humoral (i.e. autoantibodies specific for thyroglobulin, thyroxine and triiodothyronine) effector mechanisms. Other suggested factors include impaired peripheral immune suppression (i.e. the malfunction of regulatory T cells) or an additional pro-inflammatory effect of T helper 17 lymphocytes. The aim of this study was to evaluate immunological changes in canine hypothyroidism. Twenty-eight clinically healthy dogs, 25 hypothyroid dogs without thyroglobulin antibodies and eight hypothyroid dogs with these autoantibodies were enrolled into the study. There were alterations in serum proteins in hypothyroid dogs compared with healthy controls (i.e. raised concentrations of α-globulins, ß2- and γ-globulins) as well as higher concentration of acute phase proteins and circulating immune complexes. Hypothyroid animals had a lower CD4:CD8 ratio in peripheral blood compared with control dogs and diseased dogs also had higher expression of interferon γ (gene and protein expression) and CD28 (gene expression). Similar findings were found in both groups of hypothyroid dogs. Canine hypothyroidism is therefore characterized by systemic inflammation with dominance of a cellular immune response.

Proteome of spleen CD4 lymphocytes in mouse preimplantation pregnancy.

Pregnancy exerts profound impact on female immune system. The first signs of pregnancy recognition by immune system are observed even before implantation. The most visible effects are present in the local compartment, i.e. in uterine draining lymph nodes and the decidua, while peripheral changes are less obvious. In our recent paper we indicated that costimulation phenotype of APCs in spleens of female mice during the preimplantation period of pregnancy differs from mice in pseudopregnancy. However, the effect of differential costimulation in the context of the T lymphocyte function at periphery in early pregnancy is still unknown. For that reason, we decided to investigate global protein expression in splenic CD4(+) lymphocytes in order to identify and validate the most important biomarkers characteristic for the preimplantation period of pregnancy at periphery. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were utilized to analyze the protein expression pattern of magnetically sorted CD4(+) lymphocytes from spleens of pregnant and pseudopregnant females at 3.5 days after mating. The first goal of this study was to create a 2-DE map of the splenic CD4(+) T cells of pregnant mice. As a result, 106 protein spots from 373 were identified using MS. The comparison of lymphocyte protein patterns between pregnant and pseudopregnant mice depicted differential expression of 11 identified proteins belonging to the group of proteins involved in cytoskeletal structure, cell motility and metabolism. Profoundly diminished expression of cofilin-1, F-actin capping protein subunit alpha and malate dehydrogenase proteins in lymphocytes of pregnant mice indicates that preimplantation pregnancy could change the activation state of peripheral CD4(+) lymphocytes.

Accidental discovery of Trypanosoma theileri in the in vitro culture of the heifer lymphocytes.

The diagnostics of the Trypanosoma sp. invasion by means of the classic methods i.e. the methods of thin smears or thick drop or even the microhematocrite method, especially when intensity of infection is low, is very difficult. In our climatic zone, trypanosomosis is usually considered as an exotic disease. An opportunistic model of the infection with the parasite and a lack of current data on the prevalence of T. theileri in the cattle in Poland cause that it is neglected as a potential reason of contamination of tissue cultures in cattle. We showed the presence of T. theileri in culture of isolated lymphocytes from one of six heifers examined. It seems that the prevalence of the invasion of the parasite is not very intense but it should be considered as a possible threat for bovine cell culture. It is also worth including this parasitosis in the differential diagnostics of other diseases that are infectious and/or proceed with symptoms of immunosuppression.

Pleiotropy and redundancy of STAT proteins in early pregnancy.

Signal transducers and activators of transcription (STATs) are a group of proteins involved in signal transduction from numerous bioactive substances. Hormones and cytokines such as leukaemia inhibitory factor, interferon-tau and prolactin, which play key roles during early pregnancy, activate the Janus kinase (JAK)/STAT signalling pathway. The STATs are thus involved in the regulation of implantation, establishing uterine receptivity and regulation of the maternal immune response. It seems that STATs can orchestrate signals from hormones and cytokines in different cell types and may therefore generate numerous biological effects, despite the relatively small number of receptors activating the JAK/STAT pathway. This review summarizes the participation of STATs in the main processes of early pregnancy, especially regarding their pleiotropy and redundancy.

Differential effect of IFN-tau on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers.

IFN-tau is a signaling protein secreted by the bovine conceptus during the peri-implantation period and responsible for pregnancy recognition. Its main role is the prevention of pulsatile release of luteolytic PGF2alpha, but it also exerts immunomodulatory activities characteristic for other type I interferons. The aim of the study was to examine the effect of IFN-tau on the proliferation and distribution of peripheral blood lymphocyte subsets during one-way mixed lymphocyte reaction (MLR) in cows and heifers. IFN-tau inhibited the proliferative response of lymphocytes in MLR both in cows and heifers in a dose-dependent manner, but cow lymphocytes were less susceptible than those ones from heifers. It was also showed that IFN-tau differentially changed lymphocyte subsets distribution in MLR in cows and heifers. In cows, the relative percentage of CD8(+) cells after MRL in the presence of IFN-tau was significantly lower than in heifers. Differential effect of rIFN-tau on proliferation and lymphocyte subsets distribution in a one-way MRL in cows and heifers indicated that the age of the mother is an important factor in immunomodulatory effect towards developing bovine embryo.

The effect of Ureaplasma diversum activated mononuclear leukocytes on the development and interferon-tau production by bovine IVF-derived embryos.

Ureaplasma diversum is an opportunistic pathogen of the bovine genital tract causing herd outbreaks of granular vulvitis, abortion and infertility. Early embryonic death probably contributes to reduction of the reproductive performance in cows, however, pathogenesis of the disease remains obscure. The aim of the study was to examine whether activation of mononuclear leukocytes by U. diversum may affect embryo development and IFN-tau production. Bovine peripheral blood mononuclear leukocytes were cultured with U.diversum antigen for 24 h. The levels of IL-1, TNF-alpha, NO and GM-CSF in the cell culture supernatants were measured. IVF-derived embryos were cultured in the presence of supernatants from activated leukocytes. The development of embryos until day 6 postinsemination and the rate of morulae/blastocysts were determined. IFN-tau production in supernatants of cultured embryos was examined by inhibition of a virally-induced cytopathic effect. The results showed that U. diversum stimulated mononuclear leukocyte production of IL-1, TNF-alpha and NO. Supernatants from U. diversum-activated cells did not impair the rates of the embryo development and blastocyst formation. The products of activated leukocytes increased the IFN-tau production by cultured blastocysts. This suggest that U. diversum infection provides leukocyte-mediated signals for developing embryos for generation of additional production of cytokine - an important component of innate immunity.

Differential effects of heparin on NO and tumor necrosis factor-alpha production in bovine blood mononuclear cells stimulated with Salmonella typhimurium lipopolisaccharide.

We investigated the influence of heparin, one of the extracellular matrix (ECM) components, on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by bovine peripheral blood mononuclear cells (PBMC) and monocytes left to adhere for 2 (freshly adherent monocytes) and 48 h (resting monocytes), activated with Salmonella typhimurium lipopolysaccharide (LPS). After 24-h stimulation with LPS, heparin (100 microg/ml) increased (by about 40%) NO production by peripheral blood mononuclear cells and by freshly adherent monocytes. However, it did not change NO synthesis by the resting monocytes. Unlike its influence on NO level, heparin diminished TNF-alpha production by PBMC and monocytes stimulated with LPS. Microscopical examination of PBMC stained with biotin-labeled heparin, showed that both lymphocytes and monocytes were able to bind this glycosaminoglycan. We suggest that heparin, as a component of ECM, modulates the early response of monocytes to exogenous stimuli.

[IL-6 and TNF-alpha levels in oviductal epithelial cells culture after escherichia coli LPS stimulation]. / IL-6 i TNF-alpha w hodowlach blony sluzowej jajowodów po stymulacji lipopolisacharydem (LPS) Escherichia coli.

UNLABELLED: The aim of the study was to determine wether Gram-negative bacterial cell membrane lipopolysaccharides (endotoxine) can change the IL-6 and TNF-alpha cytokine concentration synthetized by fallopian tube endothelial cells. For the study 5 normal fallopian tubes from females at their reproductive age who underwent total hysterectomy due to uterine myoma were used. The fallopian tubes specimens (endothelial tissue) 2 mm2 fixed in 0.5 ml DMEM/HAM F-12 (GibcoBRL) solution with 15% FCS, Gentamycin, Fungizone, ITS (GibcoBRL) were incubated at 37 degrees C with 5% CO2. The explants were stimulated by Escherichia coli lipopolysaccharide (LPS) at ascending concentrations 1 ng/mL, 10 ng/mL and 100 ng LPS/mL incubation media. Tissue specimen incubated in a media without LPS were used for control test. IL-6 activity in the supernatants were determined by Van Sinc method, TNF-alpha activity were determined against WEHI-164.13. cells according to Espevik and Nisser-Mayer. The presence of TNF-alpha and IL-6 were confirmed in all the supernatants of the incubated fallopian tubes explants. The LPS stimulation caused a concentration increase in both cytokines. The maximum cytokine concentration level was observed in the incubation stimulated at 1 and 10 ng LPS/mL incubation media. The use of the highest LPS concentration retarded the cytokine production. CONCLUSION: The TNF-alpha and IL-6 cytokines dose-dependent production is caused by the fallopian tubes LPS stimulation.

Effect of reactive oxygen species and the activity of antioxidant systems on human semen; association with male infertility.

A range of compounds with a role in oxidative stress were measured in ejaculates from 40 normozoospermic individuals and 93 infertile males. Ejaculates were classified according to WHO criteria. Seminal plasma and the sperm cell fraction were assessed separately for superoxide dismutase (SOD), catalase, xanthine oxidase, capability for singlet oxygen trapping and content of thiobarbituric acid-reactive substances (TBARS). Pathological cases defined as oligozoospermia, asthenozoospermia, or teratozoospermia revealed different backgrounds of oxidative stress as reflected by different levels of tested substances in every type of sperm pathology. In the majority of abnormal ejaculates, a significant increase in intracellular activity of SOD, decreased intracellular levels of catalase, elevated levels of xanthine oxidase and TBARS, and severely impaired singlet oxygen trapping were observed when compared to normozoospermic ejaculates. Interrelationships between SOD and TBARS, and between xanthine oxidase and catalase, appeared to be of key importance when analysed separately in seminal plasma and in spermatozoa or in a combination of both. Elevated xanthine oxidase levels and low capacity for singlet oxygen trapping are statistically significant factors for the evaluation of male infertility which can develop as a result of persistent oxidative stress.

Ureaplasma diversum as a cause of reproductive disease in cattle.

This article includes a brief review of the classification, habitat, and characteristics of the ureaplasmas, followed by a discussion of the pathogenesis, transmission, clinical syndromes, diagnosis, immunity, and treatment of Ureaplasma diversum infections in cattle.

Activation of murine macrophages and lymphocytes by Ureaplasma diversum.

Ureaplasma diversum is a pathogen in the bovine reproductive tract. The objective of the research was to study interactions with macrophages and lymphocytes which might elucidate aspects of pathogenetic mechanisms of this organism. We studied the activation of murine macrophages of C3H/HeN (LPS-responder) and C3H/HeJ (LPS-low-responder) genotype for TNF-alpha, IL-6, IL-1 and nitric oxide production and blastogenic response of C3H/HeJ splenocytes after Ureaplasma diversum stimulation. Live and heat-killed U. diversum induced TNF-alpha, IL-6 and IL-1 in peritoneal macrophage cultures of both C3H/HeN and C3H/HeJ mice in a dose dependent manner. Interferon-gamma modulated the cytokine production, by increasing the production of TNF-alpha, IL-6 and nitric oxide, but IL-1 secretion was only enhanced in C3H/HeJ macrophages stimulated by live ureaplasmas. Supernatant of U. diversum sonicate was mitogenic for murine spleen lymphocytes. The blastogenic response was dose dependent, and stimulation with both U. diversum and Concanavalin A seemed to have an additive effect. These results suggest that U. diversum, similar to other mycoplasmas, activates murine macrophages and lymphoid cells. The studies should be repeated with bovine cells in order to elucidate pathogenetic aspects of inflammation in cattle caused by U. diversum.